The Notch receptor family is a class of evolutionarily conserved transmembrane receptors that transmit signals affecting development in organisms as diverse as sea urchins and humans. Notch receptors and their ligands Delta and Serrate (known as Jagged in mammals) are transmembrane proteins with large extracellular domains that contain epidermal growth factor (EGF)-like repeats. The number of Notch paralogues differs between species. For example, there are four Notch receptors in mammals (Notch1-Notch4), two in Caenorhabditis elegans (LIN-12 and GLP-1) and one in Drosophila melanogaster (Notch). Notch receptors are proteolytically processed during transport to the cell surface by a furin-like protease at a site S1, which is N-terminal to the transmembrane domain, producing an extracellular Notch (ECN) subunit and a Notch transmembrane subunit (NTM). These two subunits remain non-covalently associated and constitute the mature heterodimeric cell-surface receptor.
Notch1 ECN subunits contain 36 N-terminal EGF-like repeats followed by three tandemly repeated Lin 12/Notch Repeat (LNR) modules that precede the S1 site. Notch3 ECN has a similar structure, but with 34 EGF-like repeats. Each LNR module contains three disulfide bonds and a group of conserved acidic and polar residues predicted to coordinate a calcium ion. Within the EGF repeat region lie binding sites for the activating ligands. The Notch1 and Notch3 NTMs comprises an extracellular region (which harbors the S2 cleavage site), a transmembrane segment (which harbors the S3 cleavage site), and a large intracellular region (ICN or ICD) that includes a RAM domain, ankyrin repeats, a transactivation domain and a carboxy-terminal PEST domain. Stable association of the ECN and NTM subunits depends upon a heterodimerization domain (HD) comprising the carboxy-terminal end of the ECN (termed HD-N) and the extracellular amino-terminal end of NTM (termed HD-C). Before ligand-induced activation, Notch is maintained in a resting conformation by a negative regulatory region (NRR), which comprises the three LNRs and the HD domain.
Binding of a Notch ligand to the ECN subunit initiates two successive proteolytic cleavages that occur through regulated intramembrane proteolysis. The first cleavage by a metalloprotease (ADAM17) at site S2 renders the Notch transmembrane subunit susceptible to the second cleavage at site S3 close to the inner leaflet of the plasma membrane. Site S3 cleavage, which is catalyzed by a multiprotein complex containing presenilin and nicastrin and promoting γ-secretase activity, liberates the intracellular portion of the Notch transmembrane subunit, allowing it to translocate to the nucleus and activate transcription of target genes. (For review of the proteolytic cleavage of Notch, see, e.g., Sisodia et al., Nat. Rev. Neurosci. 3:281-290, 2002.)
Five Notch ligands of the Jagged and Delta-like classes have been identified in humans (Jagged1 (also termed Serrate1), Jagged2 (also termed Serrate2), Delta-like1 (also termed DLL1), Delta-like3 (also termed DLL3), and Delta-like4 (also termed DLL4)). Each of the ligands is a single-pass transmembrane protein with a conserved N-terminal Delta, Serrate, LAG-2 (DSL) motif essential for binding Notch. A series of EGF-like modules C-terminal to the DSL motif precede the membrane-spanning segment. Unlike the Notch receptors, the ligands have short cytoplasmic tails of 70-215 amino acids at the C-terminus. In addition, other types of ligands have been reported (e.g., DNER, NB3, and F3/Contactin). (For review of Notch ligands and ligand-mediated Notch activation, see, e.g., D'Souza et al., Oncogene 27:5148-5167, 2008.)
The Notch pathway functions during diverse developmental and physiological processes including those affecting neurogenesis in flies and vertebrates. In general, Notch signaling is involved in lateral inhibition, lineage decisions, and the establishment of boundaries between groups of cells. (See, e.g., Bray, Mol. Cell Biol. 7:678-679, 2006.) A variety of human diseases, including cancers and neurodegenerative disorders have been shown to result from mutations in genes encoding Notch receptors or their ligands. (See, e.g., Nam et al., Curr. Opin. Chem. Biol. 6:501-509, 2002.)
The role of Notch1 as an oncoprotein was demonstrated in leukemia involving T-cell progenitors. This role was first recognized in human acute lymphoblastic leukemia (T-ALL). (See, e.g., Aster et al., Annu. Rev. Pathol. Mech. Dis. 3:587-613, 2008.) T-ALL is an aggressive leukemia that preferentially afflicts children and adolescents. A recurrent t(7; 9)(q34; q34.3) chromosomal translocation, which creates a truncated, constitutively active variant of human Notch1, was identified in a subset of T-ALLs. In addition to the (7; 9) translocation, frequent gain-of-function mutations in human Notch1 were later discovered in more than 50% of all human T-ALLs. (See Weng et al., Science, 306:269-271, 2004.) Those mutations occur in the extracellular HD domain and the intracellular PEST domain. Other studies showed that retroviral-based expression of Notch1 ICN in bone marrow cells caused T-ALL in mouse models that received the transplanted bone marrow cells. (See Aster et al., Mol. Cell Biol. 20:7505-7515, 2000.)
Consistent with this role for Notch1 in leukemia involving T cell progenitors, Notch1 signaling has been shown to be essential for T cell development in mouse models, and Notch1-mediated signals promote T cell development at the expense of B cell development. (See, e.g., Wilson et al., J. Exp. Med. 194:1003-1012, 2001.) Further roles for Notch1 in leukemia have been described. Activating mutations in the Notch1 PEST domain have been reported at low frequency in human acute myeloid leukemia (AML) and in lineage switch leukemias, suggesting that activating mutations in Notch1 may occur in a leukemic stem cell that precedes myeloid and T-lineage commitment. (See Palomero et al., Leukemia 20:1963-1966, 2006.)
Prior to the discovery of the frequent Notch1 gain-of-function mutations in T-ALL, it was observed that enforced expression of Notch3 ICN in the thymus caused T-cell leukemia/lymphoma in transgenic mice. (See Bellavia et al., EMBO J. 19:3337-3348, 2000.) Notch3 mRNA was also reported as being expressed in all of thirty T-ALL patient samples analyzed, whereas it was not detected in normal peripheral blood T lymphocytes and non-T cell leukemias. (See Bellavia et al., Proc. Nat'l Acad. Sci. USA 99:3788-3793, 2002.)
Notch1 and Notch3 are also associated with a variety of other cancers. For instance, in solid tumors, increased Notch1 expression has been observed in human cancers of the cervix, colon, lung, pancreas, skin, and brain (see, e.g., Leong et al., Blood 107:2223-2233, 2006), and elevated expression of Notch1 is correlated with poor outcome in breast cancer (see, e.g., Parr et al., Int. J. Mol. Med. 14:779-786, 2004; Reedijk et al., Cancer Res. 65:8530-8537, 2005). A chromosomal translocation (15; 19) has been identified in a subset of non-small cell lung tumors, and the translocation is thought to elevate Notch3 transcription. In ovarian cancer, Notch3 gene amplification was found to occur in ˜19% of tumors, and overexpression of Notch3 was found in more than half of ovarian serous carcinomas. Overexpression of activated Notch1 and Notch3 in transgenic mice induces mouse breast tumors, and overexpression of Notch3 is sufficient to induce choroid plexus tumor formation in a mouse model, suggesting a role for Notch3 in the development of certain brain tumors. (For review of Notch3 in cancer, see Shih et al. Cancer Res. 67:1879-1882, 2007.)
Certain anti-Notch1 antagonist antibodies having therapeutic efficacy have been described. (See U.S. Patent Application Publication No. US 2009/0081238 A1, expressly incorporated by reference in its entirety herein.) For example, such antibodies bind to the negative regulatory region (NRR) of Notch1, block Notch1 signaling, disrupt angiogenesis and vascularization, and inhibit tumor growth in mouse xenograft models of non small cell lung carcinoma and colon adenocarcinoma. Certain antibodies described therein bind to LNR-A and LNR-B (the first and second of the three LIN12/Notch Repeats) and HD-C of Notch1 NRR. Other anti-Notch1 antibodies that bind to the EGF repeat region of Notch1 and block Notch1 activity, perhaps by blocking ligand binding, have also been described. (See International Publication No. WO 2008/091641.)
Certain anti-Notch3 antagonist antibodies have also been described. (See U.S. Patent Application Publication No. US 2008/0226621 A1, expressly incorporated by reference in its entirety herein.) Such antibodies bind to the negative regulatory region (NRR) of Notch3 and block Notch3 signaling. Certain antibodies described therein bind to LNR-A (the first of the three LIN12/Notch Repeats) and HD-C (referred to alternatively as the second dimerization domain in US 2008/0226621 A1) of Notch3 NRR. Other anti-Notch3 antibodies that bind to the EGF-like repeat region of Notch3 and block Notch3 activity, perhaps by blocking ligand binding, have also been described. (See Li et al., J. Biol. Chem. 283:8046-8054, 2008.)
Gamma-secretase inhibitors (GSIs), which are pan-Notch inhibitors that inhibit multiple Notch receptors, have been proposed for treatment of Notch-related diseases, and in fact have been used in clinical trials for the treatment of T-ALL. (See Roy et al., Curr. Opin. Genet. Dev. 17:52-59, 2007; Deangelo et al., J. Clin. Oncol. 2006 ASCO Annual Meeting Proceedings Part I 24:6586, 2006.) However, GSIs cause weight loss and intestinal goblet cell metaplasia, reflecting the role that Notch plays in determining cell fate by maintaining proliferation of intestinal crypt progenitor cells and prohibiting differentiation to a secretory cell fate. (See van Es et al., Nature 435:959-963, 2005). Although these side effects of pan-Notch inhibition may be manageable in a clinical setting, inhibitors that target individual Notch receptors, and therefore minimize or reduce these side effects, may be advantageous.
There is a need in the art for further therapeutic methods of treating cancer by targeting Notch receptors. The invention described herein meets the above-described needs and provides other benefits.